1. Field of the Invention
This invention relates generally to fluorescence polarization immunoassays and reagents useful therein, and particularly to such an assay for the analyte digoxin in a biological fluid, wherein the assay is improved by the use of a novel precipitation reagent which is effective to extract both the analyte and to precipitate interfering proteins.
2. Background Art
Digoxin is a potent cardiac glycoside widely prescribed for the treatment of patients suffering from congestive heart failure, as well as some types of cardiac arrhythmias. Digoxin intoxication is a common and serious problem in the clinical setting. This is, in part, because cardiac glycosides have a low therapeutic ratio (a very small difference between therapeutic and toxic tissue levels). Coupled with the low therapeutic ratio is a marked patient variability in response to the same dosage of drug, resulting in often unpredictable drug serum levels. Intoxication symptoms are often indistinguishable from the original condition for which the drug was prescribed, and it may not be readily apparent whether the patient has been under- or over-dosed. Monitoring of serum or plasma digoxin levels combined with other clinical data can provide the physician with useful information to aid in adjusting patient dosage, achieving optimal therapeutic effect while avoiding useless subtherapeutic or harmful toxic dosage levels.
Conventional monitoring of digoxin levels in biological fluids such as serum, plasma, urine, spinal and amniotic fluid and the like, has typically depended on commercially available radioimmunoassays (RIA) and nonisotopic assays such as fluorescence polarization immunoassays (FPIA). Certain methodological problems with various commercially-available RIA for digoxin have produced inconsistencies in results from these methods. It has been theorized that variations in the albumin concentration of individual patient serum or plasma samples and in the digoxin standards used, as well as the presence of digoxin-like immunoreactive and/or other presently unidentified substances (especially in patients with renal failure), may account for discrepencies observed between different types of RIA (See W. H. Porter, et al., Effect of Protein Concentration on the Determination of Digoxin in Serum by Fluorescence Polarization Immunoassay, Clin. Chem. 30/11, 1826-1829 (1984)).
The use of FPIA for the measurement of therapeutic drugs and other analytes of interest in the foregoing biological fluids is well known in the art. In particular, the advent of FPIA for measurement of human serum and plasma digoxin has enabled significant advantages to be achieved in analysis of this drug over RIA, most importantly in terms of reagent stability, user convenience and speed of analysis. However, investigators have reported that falsely low or high digoxin values may be found with conventional FPIA when the protein concentration of the test sample is abnormally low or high, thus constituting a potential limitation upon such assays when they are used for routine monitoring of digoxin in the laboratory setting (Porter, et al., supra).
One FPIA for serum or plasma digoxin, commercially available from Abbott Laboratories, Abbott Park, Ill. under the registered trademark TDx, utilizes a pretreatment of the sample with substantially an equal volume of 5% trichloroacetic acid solution in water (Abbott Code 9511-30) to precipitate proteins from the sample, thus minimizing interference produced by native background fluorescence of the proteins and improving the signal-to-noise ratio of the assay; e.g., when the assay is carried out using an instrument such as the TDx Fluorescence Polarization Analyzer, also commercially available from Abbott Laboratories. Results of studies of serum digoxin levels measured by this TDx FPIA generally have been favorably compared to those obtained from conventional RIA (Porter, et al., supra). However, these same studies sometimes found that the mean concentrations of digoxin by FPIA generally appeared lower by about 10 to 14 percent, when compared to RIA results. It has been suggested (Porter, et al., supra) that the source of this difference may be that digoxin is bound to the protein precipitate during the pretreatment step, the magnitude of the binding being related to the protein concentration. Therefore, differences between the protein concentration in the calibrators for FPIA (50 grams/liter) and normal serum (60-80 grams/liter) would largely account for low results for digoxin by FPIA. Subsequent research by the present inventors has revealed that approximately 30% of digoxin in typical serum samples is protein-bound. Accordingly, in conventional digoxin FPIA, about 30% of this analyte is coprecipitated, during pretreatment, with the serum proteins, yielding an assay value below that of the actual digoxin present.